Esponjas do gênero Dysidea (Ordem: Dictyoceratida) caracterizam-se por apresentarem grande diversidade de metabólitos secundários, muitos dos quais apresentam potentes atividades biológicas. Este trabalho descreve o estudo de duas amostras de esponjas da espécie Dysidea robusta, DR1 e DR2, coletadas no litoral da Bahia em 1999. Tal estudo consistiu no fracionamento das amostras, nas análises de seus extratos brutos por LC-MS e técnicas de RMN- mono e bidimensionais. Dentre os extratos de DR1, a fração DR1-EP-5A obtida do extrato éter de petróleo apresentou uma mistura de três ceramidas saturadas (22, 23 e 24). Já da amostra DR2, as frações do extrato aquoso DR2-AQ-6B e DR2-AQ-6D mostraram ser constituídas por derivados do ácido pirodisinóico (18, 19, 20 e 21). Com exceção do ácido pirodisinóico (18), os demais compostos isolados ainda não foram relatados na literatura. / Sponges of the genus Dysidea (Order: Dyctioceratida) are characterized as sources of several biologically active secondary metabolites. This work describes the study of two samples of D.robusta, DR1 and DR2, both collected at the Bahia state coastline, in 1999. The investigation aimed the crude extract fractionation and analysis by LC-MS and by 1D and 2D NMR techniques. Among the extracts DR1, the fraction DR1-EP-5A obtained from the petroleum ether extract showed a mixture of three saturated ceramides, represented by 22, 23 and 24. From the DR2 sample, the fractions obtained from the aqueous extract DR2-AQ-6B and -6D presented pirodisinoic acid derivates 18, 19, 20 and 21. Except for pyrodisinoic acid (18), all other isolated compounds haven´t been reported in the literature yet.

Orientador: Alberto José Cavalheiro / Banca: Cíntia Duarte de Freitas Milagre / Banca: Marcelo Telascrêa / Resumo: As 5,6-diidro-2-pironas 6-substituídas, também conhecidas como estirilpironas, são uma importante classe de metabólitos secundários presentes em plantas. São substâncias biologicamente ativas, apresentando como, por exemplo, atividade anticâncer, antioxidante, antifúngica e antiviral. O gênero Cryptocarya, pertence à família Lauraceae e apresenta diversas estirilpironas em suas mais diversas partes: folhas, cascas, sementes e raízes. Foram selecionadas para análise as espécies C. mandioccana, C. moschata e C. botelhensis. Foram utilizadas apenas as folhas, por apresentarem facilidade para coleta e abundância. Este trabalho tem como objetivo a identificação das estirilpironas presentes em três espécies brasileiras de Cryptocarya com o uso das técnicas HPLC-DAD-MS. Foi desenvolvido método cromatográfico para análise do extrato hidroalcoólico das folhas de espécies de Cryptocarya por HPLC-DAD-MS utilizando etanol como fase orgânica na fase móvel. Com os espectros no UV foi possível identificar que as classes de metabólitos presentes nas amostras eram alcaloides, flavonoides e estirilpironas. Utilizando os dados de MS e MS/MS, foi possível a caracterização de estirilpironas presentes nos extratos, assim como a sugestão de suas fragmentações por espectrometria de massas, além da identificação dos íons m/z 163 e m/z 189 característicos da fragmentação das estirilpironas. Foi detectado o íon m/z 423 no extrato de C. moschata, referente a um possível dímero da goniotalamina, sem rela... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The 6 - substituted 5,6 - dihydro - 2 - pyrones, also known as styryl py rone s, are an important class of secondary metabolites present in plants. They are biologically active substances, presenting, for examp le, anticancer, antioxidant, antifungal and antiviral activity. The genus Cryptocarya belongs to the family Lauraceae and presents several styrylpyrones i n its most diverse parts: leaves, barks, seeds and roots. The species C. mandioccana, C. moschata and C. botelhensis were selected for an alysis. We chose to use only leaves, because they are easy to collect and abu n dant . The objective of this work is to identify the styrylpyrones present in three Brazilian Cryptocarya species using the HPLC - DAD - MS technique s. A chromatographic method was developed to analyze the hydroal coholic extract from leaves Cryptocarya species by HPLC - DAD - MS using ethanol as the organic solvent in the mobile phase. With the UV spectra were possible to identify the classes of metabolites present in the samples as alkaloids, flavonoids and styrylpyrones . Addtitionaly, with the MS and MS 2 data, was possible to characterize the styrylpyrones present in the extracts, as well as the suggestion of their fragments by mass spectrometry, in additi on to the identification of íons m/z 163 and m/z 189 characteristics of the f ragmentation of styry lpi rones. The m/ z 423 ion detected in C. moschata extract was tentatively attributed to a goniotalamine dimer, with no previuous reports for Cryptocarya species. W ith the help of purified pirones obtained by the group, it was possible to compare their retention times with the data obtained from leaf extracts of the Cryptocarya species, helping to identify the substances present. Besides the styrylpyrones, the alkaloids menisperin and xantoplanin were also identified in the three species studied. There is a great difference in the amount of... / Mestre

Rhizophytic algae are large, abundant primary producers throughout tropical and subtropical areas worldwide where they grow as an understory in seagrass beds, as well as form mixed or monospecific beds of exclusively rhizophytic algal species. In this dissertation, "rhizophytic algae" refers to coenocytic green algae (Chlorophyta) in the order Bryopsidales that use a net of rhizoids to anchor in unconsolidated sediments. In the development of seagrass beds, rhizophytic algae colonize bare patches and are thought to facilitate seagrass colonization by stabilizing sediments and providing organic matter. However, despite their prominence little is known about many aspects of the ecology of rhizophytic algae. Detailed information on the abundance and biomass of rhizophytic algae at the species level is scarce and the belowground components are seldom quantified. Moreover, rhizophytic algal communities located along the central west coast of Florida have received very little study. At three shallow coastal sites in the Lower Florida Keys and one on the central west coast of Florida, I measured the abundance, biomass, organic content, and morphometric features of the above- and belowground portions of all rhizophytic algal species present along transects in seagrass-algal bed habitat. Relatively diverse assemblages of these algae were present both in areas with and without a seagrass canopy, though dense (greater than or equal to 50%) seagrass cover correlated with decreased algal richness. Rhizophytic algal densities at Keys sites ranged from 68 - 143 thalli m-2 with total dry weights of 76.4 - 226.7 g m-2 with only calcified species present. The west coast of Florida site had the highest aboveground organic biomass (180 g m-2), the highest abundance of rhizophytic algae (365 thalli m-2), and abundant uncalcifed algae of the genus Caulerpa. Morphometric characteristics varied within a species among sites and may reflect differences in abiotic variables such as sediment grain size. The anchoring structures of these algae, made up of fine rhizoids and attached sediment, occupied up to 5.3% of the total volume of the top 5 cm of substrate. My results indicate that across rhizophytic algal species, even within a genus, the production of belowground structure and potential influence on ecosystem function is highly variable and not necessarily related to the aboveground biomass. These results provide new information on belowground structure provided by rhizophytic algal species and characterize the rhizophytic algal community on the central west coast of Florida. The role of rhizophytic algae in seagrass bed succession has been recognized, but little is known about the rate and species composition of colonization of recently created bare patches. In a series of field experiments at three sites on the central west coast of Florida, recruitment by rhizophytic algae into created cleared areas was rapid and dominated by two species of Penicillus and Udotea flabellum. In three weeks, rhizophytic algae were able to recruit, grow to their full height, and bind sufficient sediment to create full-sized holdfasts. Additional field experiments described here show thalli of all of the rhizophytic algal species tested (three species in three genera) were able to regenerate from holdfasts (with small stubs of stipe attached) in a matter of weeks. Overall, my results suggest that belowground structures play a key role in recolonization by, and recovery of, rhizophytic algae after disturbance and are likely important to the long-term persistence of these algal populations. Bryopsidalean algae often have high concentrations of defensive compounds inside their thalli and these terpenoid secondary metabolites possess anti-fouling capability in laboratory tests. Because fouling is ubiquitous in marine environments and epibonts have harmful effects on their hosts, researchers have proposed that rhizophytic algae use these compounds to prevent fouling. For this to be an effective strategy, the compounds must be presented to potential colonizers on the external aboveground surfaces. Thus, I examined the chemistry of rhizophytic algal surfaces using extractions that avoid mechanical damage. Secondary metabolites were not detected in the surface extracts of four species while these compounds were detected in the whole plant extracts. My results, coupled with previous studies on the degradation of these metabolites in seawater and the presence of fouled plants in the field, and suggest non-polar secondary metabolites are not deployed onto the surfaces of rhizophytic algae as a defense against fouling.

Submitted by Victor Alexandre Zonaro (victor.zonaro@gmail.com) on 2017-01-10T14:22:50Z No. of bitstreams: 1 Dissertação - Victor Alexadre Zonaro.pdf: 4719611 bytes, checksum: 075493217f07f41d58c2e71196b60eea (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-13T17:16:22Z (GMT) No. of bitstreams: 1 zonaro_va_me_araiq.pdf: 4719611 bytes, checksum: 075493217f07f41d58c2e71196b60eea (MD5) / Made available in DSpace on 2017-01-13T17:16:22Z (GMT). No. of bitstreams: 1 zonaro_va_me_araiq.pdf: 4719611 bytes, checksum: 075493217f07f41d58c2e71196b60eea (MD5) Previous issue date: 2016-12-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As 5,6-diidro-2-pironas 6-substituídas, também conhecidas como estirilpironas, são uma importante classe de metabólitos secundários presentes em plantas. São substâncias biologicamente ativas, apresentando como, por exemplo, atividade anticâncer, antioxidante, antifúngica e antiviral. O gênero Cryptocarya, pertence à família Lauraceae e apresenta diversas estirilpironas em suas mais diversas partes: folhas, cascas, sementes e raízes. Foram selecionadas para análise as espécies C. mandioccana, C. moschata e C. botelhensis. Foram utilizadas apenas as folhas, por apresentarem facilidade para coleta e abundância. Este trabalho tem como objetivo a identificação das estirilpironas presentes em três espécies brasileiras de Cryptocarya com o uso das técnicas HPLC-DAD-MS. Foi desenvolvido método cromatográfico para análise do extrato hidroalcoólico das folhas de espécies de Cryptocarya por HPLC-DAD-MS utilizando etanol como fase orgânica na fase móvel. Com os espectros no UV foi possível identificar que as classes de metabólitos presentes nas amostras eram alcaloides, flavonoides e estirilpironas. Utilizando os dados de MS e MS/MS, foi possível a caracterização de estirilpironas presentes nos extratos, assim como a sugestão de suas fragmentações por espectrometria de massas, além da identificação dos íons m/z 163 e m/z 189 característicos da fragmentação das estirilpironas. Foi detectado o íon m/z 423 no extrato de C. moschata, referente a um possível dímero da goniotalamina, sem relato anterior para as espécies de Cryptocarya. Com o auxilio de pironas purificadas obtidas pelo grupo, foi possível a comparação de seus tempos de retenção com os dados obtidos a partir dos extratos de folhas das espécies de Cryptocarya ajudando na identificação das substâncias presentes. Além das estirilpironas, foram identificados os alcalóides menisperina e xantoplanina nas três espécies estudadas. Nota-se uma grande diferença na quantidade de metabólitos entre as espécies analisadas, sendo que a C. mandioccana é a mais rica em estirilpironas. / The 6-substituted 5,6-dihydro-2-pyrones, also known as styrylpyrones, are an important class of secondary metabolites present in plants. They are biologically active substances, presenting, for example, anticancer, antioxidant, antifungal and antiviral activity. The genus Cryptocarya belongs to the family Lauraceae and presents several styrylpyrones in its most diverse parts: leaves, barks, seeds and roots. The species C. mandioccana, C. moschata and C. botelhensiswere selected for analysis. We chose to use only leaves, because they are easy to collect and abundant. The objective of this work is to identify the styrylpyrones present in three Brazilian Cryptocarya species using the HPLC-DAD-MS techniques. A chromatographic method was developed to analyze the hydroalcoholic extract from leavesCryptocarya species by HPLC-DAD-MS using ethanol as the organic solvent in the mobile phase. With the UV spectra were possible to identify the classes of metabolites present in the samples as alkaloids, flavonoids and styrylpyrones. Addtitionaly, with the MS and MS2 data, was possible to characterize the styrylpyrones present in the extracts, as well as the suggestion of their fragments by mass spectrometry, in addition to the identification of íons m/z 163 and m/z 189 characteristics of the fragmentation of styrylpirones. The m/z 423 ion detected in C. moschata extract was tentatively attributed to a goniotalamine dimer, with no previuous reports for Cryptocarya species. With the help of purified pirones obtained by the group, it was possible to compare their retention times with the data obtained from leaf extracts of the Cryptocarya species, helping to identify the substances present. Besides the styrylpyrones, the alkaloids menisperin and xantoplanin were also identified in the three species studied. There is a great difference in the amount of metabolites among the analyzed species, with C. mandioccana being the richest in styrylpyrones.

Submitted by (edna.saturno@ufrpe.br) on 2016-06-29T14:39:18Z No. of bitstreams: 1 Milena Dutra da Silva.pdf: 584735 bytes, checksum: 4ed04e843193f8526a523d7cedcbdb96 (MD5) / Made available in DSpace on 2016-06-29T14:39:18Z (GMT). No. of bitstreams: 1 Milena Dutra da Silva.pdf: 584735 bytes, checksum: 4ed04e843193f8526a523d7cedcbdb96 (MD5) Previous issue date: 2008-02-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Myracrodruon urundeuva Allemão (Anacardiaceae), Sideroxylon obtusifolium (Roem. & Schult.) T. D. Penn. (Sapotaceae) and Zizyphus joazeiro Mart. (Rhamnaceae) are native species of the caatinga, widely used as medicinal, specially parts from the stem bark of adult individuals. In general, it is collected hazardously and can let the plant to die and, consequently, reduce the biodiversity. This study aimed to characterize the anatomical, histochemical and phytochemical profile of these species, to the botanical certification, to identify and to localize the accumulation of the metabolites, comparing young stem and mature leaves in young and adult individuals. Usual methods in plant anatomy, phytochemical tests to detect the metabolites classes and specific phytochemical tests to phenolic compounds, iridoids and starch were made. M. urundeuva shows amphystomatic leaves with anomocytic stomata; unicellular simple and glandular trichomes, more abundant over the veins; idioblasts with prismatic crystals in the spongy parenchyma; secretory ducts associated with the phloem in the main vein of the leaf vein, in the petiole and in the stem. S. obtusifolium shows hypostomatic leaves with actinocytic stomata; tector trichomes in the leaf lamina and in the petiole; unistratified hypodermis, with idioblasts with druses; sclereids in the mesophyll; secretory ducts in the medulla of the petiole and the stem. Z. joazeiro shows anomocytic and tetracytic stomata, unistratified hypodermis, with idioblasts with druses, braciform cells in the spongy parenchyma, prismatic crystals in the main vein of the leaf, cap of gelatinous fibers in the stem. Z. joazeiro shows cumarin molecules and cinamic derivates in the stem, only in adult individuals. The other species show similar characteristics related to the metabolites in the stem and in the leaves, in young and adult individuals with different stadium of development. / Myracrodruon urundeuva Allemão (Anacardiaceae), Sideroxylon obtusifolium (Roem. & Schult.) T. D. Penn. (Sapotaceae) e Zizyphus joazeiro Mart. (Rhamnaceae) são espécies nativas da caatinga, amplamente utilizadas como medicinais, especialmente de partes do caule de plantas adultas. Em sua grande maioria, eles são coletados de forma danosa, podendo ocasionar a morte do vegetal e, por conseguinte, a perda da biodiversidade. Este estudo objetivou caracterizar o perfil anatômico, histoquímico e fitoquímico dessas espécies, para certificação botânica, identificação e localização de acúmulo de metabólitos, comparando partes jovens de caule e folhas maduras em indivíduos jovens e adultos. Foram utilizados métodos usuais em anatomia vegetal, testes fitoquímicos para detecção das classes de metabólitos e testes histoquímicos específicos para compostos fenólicos, triterpenos e amido. M. urundeuva apresenta folhas anfiestomáticas com estômatos anomocíticos; tricomas unicelulares simples e glandulares, em maior quantidade sobre as nervuras; idioblastos com cristais prismáticos no parênquima esponjoso; ductos secretores associados ao floema na nervura principal da lâmina foliar, no pecíolo e no caule. S. obtusifolium apresenta folhas hipoestomáticas com estômatos actinocíticos; tricomas tectores na lâmina foliar e no pecíolo; hipoderme uniestratificada, com idioblastos contendo drusas; esclereídeos no mesofilo; ductos de secreção na região medular do pecíolo e do caule. Z. joazeiro apresenta estômatos anomocíticos e tetracíticos, hipoderme uniestratificada, com idioblastos contendo drusas, células do esponjosobraciformes, cristais prismáticos na nervura principal da folha, calotas de fibras gelatinosas no caule. Z. joazeiro apresentou moléculas de cumarinas e derivados cinâmicos no caule, apenas na planta adulta, as demais espécies apresentaram características comuns quanto à presença dos metabólitos e sua localização no caule e nas folhas, em indivíduos jovens e adultos. Isto indica que os metabólitos ocorrem em indivíduos com graus de desenvolvimento variado.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-22T12:40:38Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Nataliane Marques de Vasconcelos.pdf: 1186432 bytes, checksum: 7aaaefe15061c83ecc86656db0d731f1 (MD5) / Made available in DSpace on 2016-07-22T12:40:38Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Nataliane Marques de Vasconcelos.pdf: 1186432 bytes, checksum: 7aaaefe15061c83ecc86656db0d731f1 (MD5) Previous issue date: 2016-02-26 / CNPq / A resistência microbiológica aos antibióticos constitui uma série problemas de saúde pública por dificultar o tratamento das infecções. As actinobactérias são fontes importantes para a descobertas de novas moléculas com atividades biológicas. A este grupo, o gênero Streptomyces possuem dentre outros, dois grupos de enzimas multimoduladoras conhecidas como policetídeo sintase (PKS) e peptídeo sintase não ribossomal (NRPS), genes relacionados com a produção de metabólitos secundários. O presente trabalho teve como objetivo investigar o potencial in vitro de metabólitos bioativos produzidos por Actinobactérias isoladas do bioma Caatinga com atividade contra diferentes isolados clínicos de Candida spp. Após ensaio primário das 45 actinobactérias apenas a linhagem PR- 32 apresentou atividade contra Candida spp, com halos de até 20 mm no meio ISP2. Posteriormente, essa linhagem foi cultivada em seis diferentes meios de cultura sendo observada melhor produção do metabólito secundário no meio 400 em 48 horas (h) de fermentação. A determinação da concentração mínima inibitória (CMI) foi determinada a partir do extrato etanólico da biomassa de PR- 32 em pH 7.0 e foi evidenciada uma CMI entre 31,25 μg/mL a 3,9 μg/mL para as leveduras testadas. A cinética de morte reforçou o resultado da CMI e mostrou que no período de 4-8 h o extrato inibiu as cepas de Candida spp. A caracterização da actinobactéria foi identificada por metodologias clássicas e pela pesquisa do gene 16S rRNA como Streptomyces sp. PR- 32. Os resultados desta caracterização sugerem uma possível espécie nova, contudo outras análises ainda precisam ser realizadas. Foi evidenciada a presença do gene nrps com aproximadamente 750 kb. Diante destes resultados podemos concluir que Streptomyces sp. PR- 32 é um isolado promissor para produção de compostos antifúngicos, sendo possível sugerir que a atividade biológica deste metabólito secundário é regulado por peptídeo sintase não ribossomal (NRPS). / The microbial resistance to antibiotics is a series of public health problems for hindering the treatment of infections. The actinomycetes are important sources for new molecules with biological activities discovered. In this group, the genus Streptomyces have among others, multimoduladoras two groups of enzymes known as polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS), genes involved in production of secondary metabolites. This study aimed to investigate the potential in vitro bioactive metabolites produced by isolated Actinobacteria biome Caatinga with activity against different clinical isolates of Candida spp. After screening of actinomycetes in 45 primary test only the PR- 32 strain showed activity against Candida spp, with halos of up to 20 mm in the middle ISP2. Subsequently, this strain was grown in six different culture media is best seen in secondary metabolite production means 400 at 48 h of fermentation. The determination of the minimum inhibitory concentration (MIC) was determined from the ethanolic extract of the biomass of PR- 32 at pH 7.0 and one MIC was observed between 31.25 mg / mL 3.9 mg / mL for yeast tested. The kinetics of death reinforced the result of CMI and showed that in the 4-8 hour period the extract inhibited the strains of Candida spp. The characterization of actinobacteria was identified by classical methods and research 16S rRNA gene as Streptomyces sp. PR- 32. The results of this characterization suggests a possible new species, but other tests that must be performed. the presence of the NRPS gene of approximately 750 kb was observed. From these results we conclude that Streptomyces sp. PR- 32 is a promising isolated to produce antifungal compounds, it is possible to suggest that the biological activity of this secondary metabolite is regulated by peptide synthase not ribosomal (NRPS).

Esponjas do gênero Dysidea (Ordem: Dictyoceratida) caracterizam-se por apresentarem grande diversidade de metabólitos secundários, muitos dos quais apresentam potentes atividades biológicas. Este trabalho descreve o estudo de duas amostras de esponjas da espécie Dysidea robusta, DR1 e DR2, coletadas no litoral da Bahia em 1999. Tal estudo consistiu no fracionamento das amostras, nas análises de seus extratos brutos por LC-MS e técnicas de RMN- mono e bidimensionais. Dentre os extratos de DR1, a fração DR1-EP-5A obtida do extrato éter de petróleo apresentou uma mistura de três ceramidas saturadas (22, 23 e 24). Já da amostra DR2, as frações do extrato aquoso DR2-AQ-6B e DR2-AQ-6D mostraram ser constituídas por derivados do ácido pirodisinóico (18, 19, 20 e 21). Com exceção do ácido pirodisinóico (18), os demais compostos isolados ainda não foram relatados na literatura. / Sponges of the genus Dysidea (Order: Dyctioceratida) are characterized as sources of several biologically active secondary metabolites. This work describes the study of two samples of D.robusta, DR1 and DR2, both collected at the Bahia state coastline, in 1999. The investigation aimed the crude extract fractionation and analysis by LC-MS and by 1D and 2D NMR techniques. Among the extracts DR1, the fraction DR1-EP-5A obtained from the petroleum ether extract showed a mixture of three saturated ceramides, represented by 22, 23 and 24. From the DR2 sample, the fractions obtained from the aqueous extract DR2-AQ-6B and -6D presented pirodisinoic acid derivates 18, 19, 20 and 21. Except for pyrodisinoic acid (18), all other isolated compounds haven´t been reported in the literature yet.

[EN] Plant viruses are obligate intracellular parasites which were used to develop recombinant plant virus vectors to express heterologous proteins and to modify endogenous metabolic pathways of natural products in plants. The main limitation of many plant virus-based systems is the difficulty to co-express various heterologous proteins in the same cell with proper subcellular localization, which is a crucial question in metabolic engineering. This work provides a solution to overcome this problem by using a potyvirus-based vector system. Potyviruses (genus Potyvirus, family Potyviridae) are plus-strand single-stranded RNA viruses, which have a genome expression strategy that allows the equimolar production of most viral proteins. On the basis of an infectious clone of Tobacco etch virus (TEV), Bedoya et al. (2010) developed an expression system in which the RNA-dependent RNA polymerase (NIb) gene was replaced by an expression cassette, harboring several heterologous proteins. This viral vector was able to express three fluorescent proteins with nucleocytoplasmic localization in equimolar amounts in transgenic tobacco plants in which NIb was supplemented in trans. Despite of the apparent simplicity of potyvirus genome expression strategy, foreign cDNA insertion is a complicated task. Thus, our first goal was to analyze the effect of gene insertion on TEV genome stability. As a result of this work, a novel insertion position was discovered at the amino-terminal end of the potyvirus polyprotein, which opened the possibility to explore new questions of recombinant protein expression. Since metabolic pathways are highly compartmentalized, proper subcellular targeting of enzymes is an essential task. Thus, our second objective centralized on the subcellular targeting of expressed proteins from the TEV-based viral vector. cDNAs coding for the green fluorescent protein (GFP) fused to chloroplast, nucleus and mitochondria targeting signal sequences were inserted into the newly described amino-terminal insertion position or into an internal site, replacing the NIb cistron. Our results showed that for protein delivery to chloroplasts and mitochondria, foreign genes have to be inserted at the amino-terminal site of the viral vector, but for nuclear delivery, both insertion positions are suitable. The last objective of this work was to investigate whether the potyvirus-based vector was able to express an entire heterologous multistep biosynthetic pathway in plant cells. For this aim we purposed to produce lycopene, a plant pigment with health promoting properties. To do so, we inserted cDNAs coding for the enzymes of a three-step metabolic pathway of bacterial origin into the potyvirus-based vector. Infected tobacco plants developed orange symptoms indicating lycopene accumulation, which was confirmed by high-performance liquid chromatography analysis and microscopy observations. Our results also illustrated that the sole expression of Pantoea ananatis phytoene synthase, crtB, is enough to induce carotenoid accumulation, conferring yellow coloration to the infected tissue and serves as reporter system to visually track viral infection in several plant species. / [ES] Los virus de plantas son parásitos intracelulares obligados que han sido utilizados para desarrollar vectores virales y expresar proteínas heterólogas y modificar rutas metabólicas endógenas de productos naturales. La principal limitación de muchos sistemas basados en virus de plantas es la dificultad de coexpresar diversas proteínas heterólogas en la misma célula con la localización subcelular apropiada, lo cual es una cuestión crucial en ingeniería metabólica. Este trabajo presenta una solución para superar este problema mediante el uso de un vector viral basado en un potyvirus. Los potyvirus (género Potyvirus, familia Potyviridae) son virus de RNA de cadena positiva simple que tienen una estrategia de expresión génica que permite la producción de la mayoría de las proteínas virales en cantidades equimolares. Basado en un clon infeccioso del virus del grabado del tabaco (Tobacco etch virus, TEV) Bedoya et al. (2010) desarrollaron un sistema de expresión en el que el gen de la RNA polimerasa dependiente de RNA (NIb) fue sustituido por un casete de expresión, que albergaba varias proteínas heterólogas. Este vector viral fue capaz de expresar tres proteínas fluorescentes con localización nucleocitoplásmica en cantidades equimolares en plantas de tabaco transgénicas que complementaban el cistron NIb en trans. A pesar de la aparente simplicidad de la estrategia de expresión génica de los potyvirus, la inserción de un cDNA foráneo es una tarea complicada. Por lo tanto, nuestro primer objetivo fue analizar el efecto de la inserción en la estabilidad del genoma de TEV. Como resultado de este trabajo, descubrimos una nueva posición de inserción en el extremo amino-terminal de la poliproteína viral que nos permitió explorar otras cuestiones sobre la expresión de proteínas recombinantes. Dado que las vías metabólicas son muy compartimentalizadas, la adecuada localización subcelular de enzimas es una tarea esencial en ingeniería metabólica. Por eso, nuestro segundo objetivo se centró en la distribución de las proteínas heterológas expresadas con el vector viral a diferentes orgánulos subcelulares. cDNAs que codificaban la proteína fluorescente verde (green fluorescent protein, GFP) fusionada a péptidos señal se insertaron en la nueva posición amino-terminal y en un sitio interno, sustituyendo el cistrón NIb, para enviarla al cloroplasto, núcleo y a la mitocondria. Nuestros resultados mostraron que para la distribución de proteínas al cloroplasto y mitocondria, los genes foráneos deben ser insertados en el sitio amino-terminal del vector viral, pero para la distribución nuclear, ambas posiciones son adecuadas. El último objetivo de este trabajo fue estudiar si el vector viral basado en potyvirus es capaz de expresar una ruta biosíntética de múltiples pasos en células vegetales. Para ello nos propusimos producir licopeno, un pigmento vegetal con propiedades beneficiosas para la salud humana. Para ello, insertamos un cDNA que codificaba las enzimas de una ruta metabólica de tres pasos de origen bacteriano en el vector viral. Las plantas de tabaco infectadas con el vector viral desarrollaron síntomas de color naranja indicando la acumulación de licopeno, que fue confirmado por análisis de cromatografía líquida de alta eficacia y observaciones de microscopía. Nuestros resultados también ilustraron que la sola expresión de la fitoeno sintasa de Pantonea ananatis, crtB, es suficiente para inducir la acumulación de carotenoides que confieren una coloración amarilla al tejido infectado y sirve como sistema reportero visual en varias especies de plantas. / [CAT] Els virus de plantes són paràsits intracel·lulars obligats que han estat utilitzats per a desenvolupar vectors virals i expressar proteïnes heteròlogues y modificar rutes metabòliques endògenes de productes naturals silenciant certs gens o expressant factors de transcripció i enzims metabòlics. La principal limitació de molts sistemes basats en virus de plantes és la dificultat de coexpressar diverses proteïnes heteròlogues en la mateixa cèl·lula amb la localització subcel·lular apropiada, cosa que és una qüestió crucial en enginyeria metabòlica. Aquest treball presenta una solució per a superar aquest problema mitjançant l'ús d'un vector viral basat en un potyvirus. Els potyvirus (gènere Potyvirus, família Potyviridae) són virus d'RNA de cadena positiva simple que tenen una estratègia d'expressió gènica que permet la producció de la majoria de les proteïnes virals en quantitats equimolars. Basat en un clon infecciós del virus del gravat del tabac (Tobacco etch virus, TEV) Bedoya et al. (2010) van desenvolupar un sistema d'expressió en el qual el gen de l'RNA polimerasa depenent d'RNA (NIb) va ser substituït per un casset d'expressió, que albergava diverses proteïnes heteròlogues. Aquest vector viral va ser capaç d'expressar tres proteïnes fluorescents amb localització nucleocitoplàsmica en quantitats equimolars en plantes de tabac transgèniques que complementaven el cistró NIb en trans. Malgrat l'aparent simplicitat de l'estratègia d'expressió gènica dels potyvirus, la inserció d'un cDNA forà és una tasca complicada. Per tant, el nostre primer objectiu va ser analitzar l'efecte de la inserció en l'estabilitat del genoma de TEV. Com a resultat d'aquest treball, hem descobert una nova posició d'inserció en l'extrem amino terminal de la poliproteïna viral que ens va permetre explorar altres qüestions sobre l'expressió de proteïnes recombinants. Atès que les vies metabòliques són molt compartimentalitzades, l'adequada localització subcel·lular d'enzims és una tasca essencial en enginyeria metabòlica. Per açò, el nostre segon objectiu es va centrar en la distribució de les proteïnes heteròlogues expressades amb el vector viral a diferents orgànuls subcelul·lars. cDNAs que codificaven la proteïna fluorescent verda (green fluorescent protein, GFP) fusionada a pèptids senyal es van inserir en la nova posició amino terminal i en un lloc intern, substituint el cistró NIb, per a enviar-la al cloroplast, nucli i al mitocondri. Els nostres resultats van mostrar que per a la distribució de proteïnes al cloroplast i mitocondri, els gens forans han de ser inserits en el lloc amino terminal del vector viral, però per a la distribució nuclear, ambdues posicions són adequades. El lloc amino terminal va resultar ser més adequat per a produir quantitats més grans de proteïnes recombinants, però el lloc d'inserció intern va demostrar ser més estable. Sobre la base d'aquests resultats, hem sigut capaços de distribuir dues proteïnes fluorescents diferents als cloroplasts i nuclis des d'un únic vector viral. L'últim objectiu d'aquest treball va ser estudiar si el vector viral basat en potyvirus és capaç d'expressar una ruta biosintètica de múltiples passos en cèl·lules vegetals. Per açò ens vam proposar produir licopè, un pigment vegetal amb propietats beneficioses per a la salut humana. Per això inserírem un cDNA que codificaba els tres enzims de una ruta metabòlica de tres passos d'origen bacterià en el vector viral. Les plantes de tabac infectades amb el vector viral van desenvolupar símptomes de color taronja indicant l'acumulació de licopè, que va ser confirmat per anàlisi de cromatografia líquida d'alta eficàcia i observacions de microscòpia. Els nostres resultats també van il·lustrar que la sola expressió de fitoè sintasa de Pantonea ananatis, crtB, és suficient per a induir l'acumulació de carotenoides que confereixen una colora / Majer, E. (2016). Metabolic engineering of plants using a disarmed potyvirus vector [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68477 / TESIS

Plant cell culture is an attractive platform technology for production and supply of important plant derived medicinals. A unique characteristic of plant cells is the ability to grow as multicellular aggregates in suspension. The presence of these non-uniform aggregates results in creation of distinct microenvironments, which can induce variations in cellular metabolism (e.g., growth, oxygen consumption and secondary metabolite synthesis). This heterogeneity can lead to unpredictable and suboptimal performance in large scale bioprocesses. One example is the Taxus cell culture system, which produces a widely used chemotherapeutic drug - paclitaxel (Taxol ®). Despite extensive process engineering efforts which have led to increased yields of paclitaxel, Taxus cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces the accumulation of paclitaxel, but to varying extents in culture. A significant negative correlation was observed between paclitaxel level and mean aggregate size of the culture, demonstrating the relevance of measuring, and potentially controlling aggregate size during long term subculture. Understanding the regulation of gene expression can provide rational engineering strategies to control variability and optimize performance of Taxus cell cultures. Biosynthetic pathway gene analyses revealed upregulation of genes upon elicitation with MeJA; results also suggested additional molecular regulatory points outside of the biosynthetic pathway. In order to fully understand Taxus molecular regulation and the relationship to paclitaxel production variability, a transcriptome-wide analysis using next generation sequencing (454 and Illumina) methods was performed. Several pathways outside of paclitaxel biosynthesis were found active upon MeJA elicitation. Global comparison of gene expression amongst cultures accumulating different levels of paclitaxel is being performed to completely understand the interactions amongst the paclitaxel biosynthetic pathway and other complimentary and competing pathways to suggest effective targets for metabolic engineering. This work collectively represents the first molecular studies to understand metabolic regulation in Taxus cell cultures. Apart from inducing paclitaxel biosynthesis, MeJA decreases cell growth in Taxus cell cultures. The MeJA-mediated repression of cell growth was shown to correlate with inhibition of cell cycle progression as evident both at the culture level through flow cytometric analyses and at the transcriptional level by repression of key cell cycle-associated genes. Results from this study provide valuable insight into the mechanisms governing MeJA perception and subsequent events leading to repression of Taxus cell growth.

The fungus Macrophomina phaseolina (Tassi) Goidanich (Mp) is the causal agent of charcoal rot in soybean and infects over 500 plant species worldwide. Mp produces various mycotoxins and is suspected of utilizing a toxin-mediated process to penetrate host tissue. Identification and evaluation of secondary metabolites produced by Mp will further elucidate the pathogenesis mechanisms used by the fungus. Mp cultures isolated from soybean were evaluated for phytotoxicity in a hydroponic soybean bioassay and chemically analyzed by LC-MS/MS. All Mp cultures at two dilutions induced phytotoxicity symptoms including chlorosis, necrosis, wilting, stunting, and death. Analysis identified 13 unreported secondary metabolites including mellein, a compound with various biological activities. The phytotoxicity of mellein was evaluated against soybean seedlings in hydroponic culture, and symptoms of wilting and stunting were observed at levels above 40 MUg/L. Observations suggest that mellein does not directly contribute to the phytotoxic effects of Mp cultures.